Neutralizing antibodies are considered the best surrogate markers of protection against a SARS-CoV-2 infection. However, the predictive value of these antibodies overlaps only slightly with other variants. In a case-control study, in pre-infected vaccinated patients, polyfunctional anti-Wuhan CD4+ T cell responses predicted protection against both the ancestral Wuhan strain and the distant Omicron variant, whereas CD4+ T cell responses to unrelated viruses (e.g., CMV, influenza) did not. Conversely, neutralizing antibodies to the Wuhan spike correlated with protection from Wuhan infection, but failed to predict breakthroughs with divergent variants such as Omicron. Confounding factors (age, sex, timing of sampling related to infection onset, number of booster doses) were accounted for to minimize bias. To explain these results, we observed very strong correlations between the anti-Wuhan CD4+ T cell responses and the corresponding anti-Omicron BA.1 CD4+ T cell response. CD4+ T cell responses may serve as a potential surrogate marker of protection, regardless of the variant.

Following the recent introduction of molecular diagnosis criteria for schwannomatosis, we decided to study the results of somatic molecular testing in patients with a tumor burden suggestive of non-NF2-related Schwannomatosis, with a particular focus on patients harboring multi-nodular schwannomas. We selected 22 patients harboring multiple peripheral nerve schwannomas, with or without associated lumbar schwannomas after a brain and spine MRI workup ruling out cranial nerve schwannomas, meningiomas and ependymomas. For each patient, at least 2 separate anatomically distinct tumor nodules were available for analysis. We distinguished mono-nodular from multi-nodular tumors and performed targeted sequencing of the NF2, SMARCB1 and LZTR1 genes for all tumors and germline DNA when available. We analyzed 69 tumor nodules in 22 patients, 19 of whom had a non-familial disease. Most patients (54

BACKGROUND:OVX033 is a potential universal sarbecovirus vaccine candidate targeting the highly conserved SARS-CoV-2 nucleocapsid (N) protein. Preclinical studies have demonstrated cross-variant protection and favorable tolerability. This first-in-human trial evaluated the safety and immunogenicity of a single intramuscular dose of OVX033 in healthy adults (18-49 years). METHODS:In this randomized, placebo-controlled, observer-blind, sequential, dose-escalation study, 48 participants, previously vaccinated with SARS-CoV-2 licensed vaccines, received either OVX033 (100µg, 250µg or 500µg; n=12 per cohort) or placebo (n=12, 4 per cohort). Solicited reactogenicity was recorded for 7 days, unsolicited adverse events (AEs) for 28 days, and serious AEs (SAEs) for 6 months. Immunogenicity assessments included anti-N IgG and N-specific T-cell responses. RESULTS:OVX033 was safe and well-tolerated whatever the dose-level. Injection site pain was the most common local reaction; fatigue, headache, and myalgia were the most frequent systemic symptoms. No severe reactogenicity, treatment-related SAEs, or safety signals were identified. OVX033 elicited a robust anamnestic anti-N IgG response, with titers peaking at Day 29, and 4-fold rises versus baseline observed in 67-100% of vaccinees. Modest increases of N-specific IFNγ CD4+ T-cell responses were measured on Days 8 and 29 after vaccination, while CD8+ T-cell responses remained below the limit of quantification. No clear OVX033 dose-effect was evidenced. CONCLUSIONS:OVX033 demonstrated a favorable safety profile and strong boosting of pre-existing N-specific humoral immunity. Although a N-specific T-cell-mediated immune response was achieved, it remained of limited amplitude. This limitation could be addressed by implementing a two-dose regimen, or through the adjuvantation of the vaccine.

Background: The increased interest in anti-platelet factor 4 (PF4)-heparin complex (anti-PF4/H) antibodies following the COVID-19 pandemic has established them as crucial players in immunothrombosis. Objectives: We aimed to investigate the involvement of anti-PF4/H antibodies during COVID-19 and after vaccination, particularly in patients with systemic inflammatory disease (SID). Methods: This retrospective study analyzed the presence of anti-PF4/H antibodies and their ability to induce platelet activation in COVID-19 patients with and without suspected heparin-induced thrombocytopenia (HIT), vaccine-induced immune thrombotic thrombocytopenia (VITT) patients, and in controls and SID patients following COVID-19 vaccination. Results: No significant increase in anti-PF4/H antibody levels was observed during COVID-19 regardless of disease severity. Despite a 2-fold increase in HIT suspicion observed during the pandemic, there was no corresponding increase in HIT diagnoses. Additionally, no significant increase in anti-PF4/H levels was noted after vaccination, even in SID patients. None of the positive anti-PF4/H antibodies detected in COVID-19 or vaccination cohorts induced platelet activation, measured by soluble P-selectin levels and flow cytometry-based on platelet microvesicle generation. Finally, in VITT patients, unlike in HIT patients, anti-PF4/H levels were strongly associated with platelet microvesicle assay and moderately with soluble P-selectin levels. Conclusion: Our study found no significant increase in anti-PF4/H antibodies in COVID-19 or after vaccination, including in SID patients. However, in VITT patients, but not in HIT patients, these antibodies were correlated with platelet activation. This finding suggests that anti-PF4/H antibodies play a different role in the pathophysiology of VITT but that their interest is limited outside clear contexts of HIT/ VITT suspicion.

Ageing is accompanied by a decline in immune function, associated with susceptibility to infections and malignancies, and reduced vaccine efficacy. These immunological changes, affect multiple components of the immune system, particularly T lymphocytes, which exhibit altered subset distributions and accumulate senescent features. CD57, a surface glycoprotein expressed on T cells, has emerged as a potential marker of terminal differentiation and senescence used for immunomonitoring in infection or cancer contexts. However, the use of CD57 as a marker of T cell senescence remains unclear. To investigate this, we analyzed CD57 expression on CD8+ and CD4+ T cells in healthy donors from two independent cohorts, considering cellular differentiation, age, cytomegalovirus status, and other senescence markers. Our findings reinforce the association between CD57 expression, T cell differentiation, and CMV seropositivity, but not with chronological age. Although CD57 is associated with altered proliferation and survival in all T cell differentiation subsets, it does not fully align with a senescent phenotype. Therefore, we propose that CD57 may be better appreciated as a marker of immunological age. Moreover, the interpretation of CD57 expression must account for CMV serostatus to avoid misleading conclusions, especially in oncology and ageing research.

BACKGROUND AND OBJECTIVES:Pain is the cardinal symptom of non-neurofibromatosis type 2 schwannomatosis (non- NF2 -SWN), and peripheral nerve schwannomas are the most frequent tumors encountered in this condition. The role of surgery in alleviating schwannoma-associated pain in schwannomatosis remains a matter of debate in the literature. METHODS:We conducted a retrospective chart review of all non- NF2 -SWN patients followed up at our Schwannomatosis Reference Center and included all patients operated from a peripheral nerve tumor. NF2 -related schwannomatosis was excluded on the basis of germline genetic study and/or absence of suggestive tumors on brain and spine MRIs. RESULTS:Fifty-nine patients were included, suffering mostly from a nonfamilial (50/59, 85%) and diffuse (40/59, 68%) non- NF2 -SWN. A germline genetic study of the NF2 , LZTR1 , and SMARCB1 genes was performed in 38 patients (64%) and demonstrated a LZTR1 gene variant in 19 cases (50%). The patients were operated from 103 peripheral nerve tumors, mainly located in major nerves (n = 67, 65%) compared with subcutaneous (n = 17, 16%) and intramuscular (n = 17, 16%) cases. Most tumors were classical discrete tumors (n = 88, 85%), while multinodular cases represented only 15% (n = 15) of cases. Pathological analysis confirmed the diagnosis of schwannoma except 2 cases of hybrid neurofibroma/schwannoma tumors. A complete resection was performed in 89% of cases with a complete relief of preoperative pain in 87% of cases. Postoperative motor and sensory deficits were encountered in 8 (8%) and 14 (14%) cases, respectively. Multinodular schwannomas were characterized by a decreased rate of complete pain relief (64% vs 90%, P = .007) and an increased rate of postoperative motor deficit (3% vs 35%, P < .001) compared with discrete tumors. CONCLUSION:Nerve-sparing surgery using intraoperative neuro-monitoring remains effective in treating pain of non- NF2 -SWN-associated peripheral nerve schwannomas, with the notable exception of multinodular tumors, also characterized by an increased rate of postoperative motor deficits.

This study aimed to assess platelet activation following implantation of the Aeson bioprosthetic total artificial heart (A-TAH). We monitored plasma levels of platelet activation markers in patients receiving A-TAH support (n = 16) throughout the follow-up period. Before implantation, soluble CD40 ligand (sCD40L) levels averaged 3,909.06 pg/ml (standard deviation [SD] = 3,772.37), remaining stable postimplantation at 3,964.56 pg/ml (SD = 2,198.85) during months 1-3 and at 3,519.27 pg/ml (SD = 1,647.04) during months 3-6. Similarly, P-selectin (sP-sel) levels were 35,235.36 pg/ml (SD = 14,940.47) before implantation, stabilizing to 33,158.96 pg/ml (SD = 9,023.11) (1-3 months) and 31,022.58 pg/ml (SD = 9,249.95) (3-6 months). Preimplantation platelet factor 4 (PF4) measured 2,593.47 ng/ml (SD = 2,167.85), remaining consistent at 2,136.10 ng/ml (SD = 1,264.47) (1-3 months) and 1,991.26 ng/ml (SD = 1,234.16) (3-6 months). Levels of neutrophil-activating peptide 2 (NAP2) were also steady, measuring 785.63 ng/ml (SD = 605.26) preimplantation, 935.10 ng/ml (SD = 517.73) at 1-3 months, and 907.21 ng/ml (SD = 501.96) at 3-6 months postimplantation. Importantly, neither aspirin nor heparin treatment affected these platelet biomarker levels. No correlation was observed between platelet activation marker levels and clinical outcomes such as pericardial effusion, nor with the timing of aspirin initiation and drain removal. Our findings confirm that A-TAH does not trigger platelet activation. The lack of association between aspirin, platelet activation, and clinical outcomes suggests the possibility of discontinuing antiplatelet therapy following A-TAH implantation in the future.

BACKGROUND:Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders. NF1 is caused by dominant loss-of-function pathogenic variants (PVs) of the tumour-suppressor gene NF1, which encodes neurofibromin, a negative regulator of rat sarcoma proteins. NF1 is an autosomal dominant disorder with complete penetrance, but a highly variable expression. Identification of genotype-phenotype correlations is challenging because of the wide clinical variability, the progressive nature of the disorder and the extreme diversity of the mutation spectrum. Only a few NF1 point variants have been associated with a specific phenotype in NF1 patients. METHODS:We investigated a large, well-phenotyped NF1 cohort. RESULTS:We report analyses of genotype-phenotype correlations in 112 NF1 patients with specific NF1 point variants: p.Arg1809 missense variants were associated with a mild form of NF1 (n=24), while a more severe phenotype was associated with codons 844-848 (n=27), p.Arg1276 (n=25) and p.Lys1423 (n=35) missense variants. We describe a new correlation for p.Arg1204 missense variants (n=11), with no neurofibroma observed in patients. Functional studies will be critical for drawing conclusions on the potential hypomorphic or dominant-negative effects of these variants. CONCLUSION:The current data confirms several genotype-phenotype correlations in NF1, which may be relevant to the management and surveillance of NF1 patients with specific NF1 PVs.

The access to next-generation sequencing, primarily performed for therapeutic purposes, has considerably increased in the real-time clinical practice in pediatric malignancies. Using germline DNA as a matched reference, this approach is also a unique opportunity to detect alterations in medically actionable genes (named "additional data"). The main objective of this project was to estimate the frequency of such genetic predisposition discoveries among children and adolescents with relapsing or refractory cancer. Among 791 children included in MAPPYACTS “MoleculAr Profiling for Pediatric and Young Adult Cancer Treatment Stratification” study, germline DNA exome sequencing was available for 674 patients with many distinct cancer types. Informed consent was obtained to disclose genetic findings if an actionable predisposition was detected. A multidisciplinary germline molecular board established the list of genes to be looked at, including 184 cancer predisposition genes with consensual surveillance guidelines, and 49 non cancer genes belonging to the ACMG Secondary Findings v3.1 list. Bioinformatic analysis included two different indel variant calling (Varscan and Haplotypecaller), quality controls and filters to ensure high-confidence variant calls, ClinVar annotations for pathological classification and in-silico function assessment by computational prediction (with several tools including CADD, SpliceAI and SPiP). Expert biologists have evaluated the pathogenicity of genetic variations with the use of genetic databases, computational predictions and medical literature. Among 184 cancer genes, 16 607 genetic variants have emerged. Twenty-five per cent of them were classified as unknown significance corresponding to a median of 7 (0;47) variants/patient and 8% of variants were retained as likely pathogenic or pathogenic variant (LPV/PV). Thus, 132 patients (19.6%) carried one LPV/PV variant among 53 cancer genes. According to inheritance patterns (we retained heterozygous variant for dominant disease and only biallelic events for recessive disease), genetic counselling was recommended for 58 patients (8.6%). Only two patients had two different cancer predisposition. The most frequently involved genes were TP53 (n=16), DICER1 (n=5), NF1 (n=4) and BRCA1 (n=4). Those identified genetic predispositions to cancer corresponded to the expected tumor spectrum in 50% of cases and were previously known in 38% of families. Furthermore, 10 patients (1.5%) were germline carriers of LPV/PV in genes involved in other genetic conditions (n=6 for cardiopathy; n=2 familial hypercholesterolemia; n=2 transthyretin amyloidosis). A specific evaluation of exome sequenced for therapeutic purposes provides germline information that could be actionable for 10.1% of families to improve cancer prevention or manage some other genetic condition. The psychological impact induced by the return of secondary findings to families will be investigated. Tiphaine Adam de Beaumais, Yahia Adnani, Léa Guerrini-Rousseau, Samuel Abbou, Cécile Acquaviva-Bourdain, Pablo Berlanga, Adeline Bonnard, Gaelle Bougeard, Franck Bourdeaut, Nelly Burnichon, Sandrine Caputo, Alain Carrié, Olivier Caron, Hélène Cavé, Albain Chansavang, Nadège Corradini, Sophie Cotteret, Philippe Denizeau, Alice Fievet, Mathilde Filser, Marion Gauthier-Villars, Birgit Geoerger, Nadim Hamzaoui, Edwige Kasper, Florence Kyndt, Ludovic Lacroix, Jessica Le Gall, Julien Masliah-Planchon, Laurence Pacot, Cécile Pagan, Mélanie Pagès, Béatrice Parfait, Eric Pasmant, Gaelle Pierron, Pascale Richard, Nathalie Roux-Buisson, Cécile Saint-Martin, Hela Sassi, Gudrun Schleiermacher, Renaud Touraine, Nancy Uhrhammer, Dominique Vidaud, Laurence Brugières, Gilles Vassal, Etienne Rouleau, Yoann Vial, Lisa Golmard, Odile Cabaret. Genetic predisposition discoveries of clinical utility by exome sequencing performed for therapeutic purposes in children with relapsing or refractory cancer in the MAPPYACTS study [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A006.

PURPOSE:No universal circulating biomarker exists for soft-tissue sarcoma (STS) and bone sarcoma. We report the translational relevance of a Droplet Digital PCR (ddPCR) assay allowing universal, specific, and dynamic detection of sarcoma-related hypermethylated ctDNA. EXPERIMENTAL DESIGN:In silico analysis (The Cancer Genome Atlas/Gene Expression Omnibus datasets, n = 8,330) identified hypermethylated DNA positions in STS/bone sarcoma, unmethylated in nonsarcoma tissues or white blood cells releasing circulating plasma cell-free DNA (cfDNA). A ddPCR assay following bisulfite conversion of cfDNA was developed. The methylation signature performances were evaluated in independent in silico cohorts (The Cancer Genome Atlas/Gene Expression Omnibus, n = 1,342). The ddPCR assay was applied to cfDNA from healthy donors, patients with metastatic STS (METASARC cohort, n = 49, 13 histotypes), and patients with STS/bone sarcoma treated with neoadjuvant chemotherapy (NEOSARC cohort, n = 42, 10 histotypes). RESULTS:A ddPCR assay targeting seven methylated genomic positions distinguished sarcoma samples from nonneoplastic mesenchymal and endothelial/liver tissues (AUC = 0.95; in silico validation set). Sensitivity allowed methylated DNA detection at a 1:1,000 dilution in genomic DNA, with a methylated allele frequency of 0.06%. ctDNA was positively detected in 45% of METASARC (22/49) and 74% of NEOSARC (31/42) patients, across all histotypes. ctDNA detection correlated with poor overall survival in METASARC patients with STS (P = 0.039). Increasing ctDNA during neoadjuvant chemotherapy was associated with poor outcomes in NEOSARC (composite criteria with poor histologic response, radiological progression, or relapse within 6 months; P = 0.0095). CONCLUSIONS:This sensitive ddPCR assay for universally methylated ctDNA enables precise detection, prognostication, and real-time monitoring of tumor burden in patients with high-grade and advanced sarcoma, regardless of histotype or origin.

BACKGROUND:Patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA) are at an increased risk for infection related to the use of immunomodulatory therapies (ITs). The objective of this study is to assess the impact of ITs on the adaptive immune responses to SARS-CoV-2. METHODS:The study population comprised 94 patients (48 SpA; 46 RA; mean age of 53±14 years) with a confirmed SARS-CoV-2 infection. 20 age-matched individuals (50±17 years), corresponding to the patients' household contacts infected at the same time, were included as the control population. Patients were stratified by treatment groups: methotrexate (MTX)/sulfasalazine (n=17/2), anti-TNF (n=24), anti-TNF+MTX (n=23), RTX (N=11), anti-IL17 (n=7) and others (n=11). The study compared the viral loads in plasma, stools and nasal swabs and the SARS-CoV-2-specific humoral and cellular immune responses (antibodies, B and T lymphocytes) following SARS-CoV-2 infection. RESULTS:Viral persistence was not observed in the blood, nasopharynx and stools of patients undergoing ITs. Overall, the SARS-CoV-2-specific humoral and T-cell responses were preserved. Patients receiving RTX showed significantly lower IgA and IgG responses to SARS-CoV-2 compared with other treatment groups. Most patients, including RTX recipients, exhibited significant CD4+T cell responses. However, RTX therapy was associated with reduced SARS-CoV-2-specific activated CD8+T cells. A correlation was observed between humoral immune parameters and CD8+ T cell activation. CONCLUSIONS:While most patients demonstrated the capacity to mount an immune response to SARS-CoV-2, treatment with RTX impacted both humoral and CD8+cell responses. Developing vaccines that elicit robust CD8+T cell responses could offer benefits to individuals undergoing ITs for inflammatory rheumatic diseases.

OBJECTIVE:We aimed to study whether the detection of circulating tumor DNA (ctDNA) may predict the risk of early relapse for patients with localized endometrial carcinoma. METHODS:Patients who underwent surgical resection at Cochin University Hospital (2021-2023) for International Federation of Gynecology and Obstetrics 2018 stage I to III endometrial carcinoma were prospectively included in a prospective biocollection cohort study. All patients had a plasma sample before surgery (EDTA collection tubes, 4-5 mL). After extraction and bisulfite-conversion of cell-free DNA, ctDNA was evaluated using a droplet-digital polymerase chain reaction assay targeting universally-hypermethylated positions in endometrial carcinoma (OXT, ZSCAN12 genes), and defined as significantly detected above the limit of detection. Patients were classified as high-risk based on 2022 European Society for Medical Oncology/European Society of Gynaecological Oncology/European Society of Pathology guidelines, or preoperative features (non-endometrioid histology, p53-abnormal tumors, or stage III). Events of interest were tumor progression or relapse (event-free survival). Adjusted-HR (aHR) was estimated using Cox regression. RESULTS:Among 128 patients included with median follow-up of 26 months (interquartile range; 15-35), ctDNA was detected in 18 patients (14%). Patients with ctDNA had a 1-year event-free rate of 67% (95% CI [48% to 92%]), vs 91% [82% to 100%] among patients without ctDNA. The ctDNA was detected in 10 (29%) patients among those with preoperative high-risk features (N = 34, 1-year event-free rate = 60% [36%-100%]). ctDNA was associated with event-free survival independently of stage (aHR = 4.26 [1.68-10.8]), 2022 guidelines high-risk (aHR = 3.72 [1.57-8.87]), or preoperative high-risk features (aHR = 3.98 [1.65-9.60]). CONCLUSIONS:Elevated ctDNA before surgery identifies a very high-risk subgroup of newly diagnosed endometrial carcinoma, suggestive of occult metastasis. Further studies are warranted to validate this finding and investigate the window of opportunity for neoadjuvant approaches.

Neurofibromatosis type -1 is a genetic disorder caused by loss -of -function variants in the tumorsuppressor NF1. Approximately 4% to 11% of neurofibromatosis type -1 patients have a NF1 locus complete deletion resulting from nonallelic homologous recombination between low copy repeats. Codeleted genes probably account for the more severe phenotype observed in NF1-deleted patients. This genotype -phenotype correlation highlights the need for a detailed molecular description. A droplet digital PCR (ddPCR) set along the NF1 locus was designed to delimitate the three recurrent NF1 deletion breakpoints. The ddPCR was tested in 121 samples from nonrelated NF1-deleted patients. Classification based on ddPCR versus multiplex ligation -dependent probe amplification (MLPA) was compared. In addition, microsatellites were analyzed to identify parental origin of deletions. ddPCR identified 77 type -1 (64%), 20 type -2 (16%), 7 type -3 (6%), and 17 atypical deletions (14%). The results were comparable with MLPA, except for three atypical deletions misclassified as type -2 using MLPA, for which the SUZ12 gene was not deleted. A significant maternal bias (25 of 30) in the origin of deletions was identified. This study proposes a fast and efficient ddPCR quantification to allow fine NF1 deletion classification. It indicates that ddPCR can be implemented easily into routine diagnosis to complement the techniques dedicated to NF1 point variant identification. This new tool may help unravel the genetic basis conditioning phenotypic variability in NF1-deleted patients and offer tailored genetic counseling. (J Mol Diagn 2024, 26: 150-157; https://doi.org/10.1016/j.jmoldx.2023.11.005)

Coordinating immune responses - humoral and cellular - is vital for protection against severe Covid-19. Our study evaluates a multicytokine CD4 + T cell signature's predictive for post-vaccinal serological and CD8 + T cell responses. A cytokine signature composed of four cytokines (IL-2, TNF- a, IP10, IL-9) excluding IFN- g, and generated through machine learning, effectively predicted the CD8 + T cell response following mRNA1273 or BNT162b2 vaccine administration. Its applicability extends to murine vaccination models, encompassing diverse immunization routes (such as intranasal) and vaccine platforms (including adjuvanted proteins). Notably, we found correlation between CD4 + T lymphocyte-produced IL-21 and the humoral response. Consequently, we propose a test that offers a rapid overview of integrated immune responses. This approach holds particular relevance for scenarios involving immunocompromised patients because they often have low cell counts (lymphopenia) or pandemics. This study also underscores the pivotal role of CD4 + T cells during a vaccine response and highlights their value in vaccine immunomonitoring.

We report our 5-year experience in neurofibromatosis type 1 prenatal diagnosis (PND): 205 PNDs in 146 women (chorionic villus biopsies, 88% or amniocentesis, 12%). The NF1 variant was present in 85 (41%) and absent in 122 (59%) fetuses. Among 205 pregnancies (207 fetuses), 135 were carried to term (119 unaffected and 16 NF1 affected children), 69 pregnancy terminations (affected fetuses), 2 miscarriages, and 1 in utero death. The majority of PND requests came from parents with sporadic NF1. We describe two PNDs in women with mosaic NF1. In both families, direct PND showed the absence of the maternal NF1 variant in the fetus. However, microsatellite markers analysis showed that the risk haplotype had been transmitted. These rare cases of germline mosaicism illustrate the pitfall of indirect PND. Our study illustrates the crucial consequences of PND for medical and genetic counseling decisions. We also point to the challenges of germline mosaics.

INTRODUCTION:The complement system is involved in numerous diseases, through diverse mechanisms and degree of activation. With the emergence of complement targeting therapeutic, simple and accessible tools to evaluate the extent of complement activation are strongly needed.METHODS:We evaluated two multiplex panels, measuring complement activation fragments (C4a, C3a, C5a, Bb, Ba, sC5b9) and intact components or regulators (C1q, C2, C3, C4, C5, FD, FP, FH, FI). The specificity of each measurement was assessed by using complement proteins depleted sera and plasma collected from patients with complement deficiencies. Normal values distribution was estimated using 124 plasma samples from healthy donors and complement activation profile was assessed in plasma collected from 31 patients with various complement-mediated disorders.RESULTS:We observed good inter-assay variation. All tested protein deficiencies were accurately detected. We established assay-specific reference values for each analyte. Except for C3, C4 and C4a, the majority of the measurements were in good agreement with references methods or published data.CONCLUSION:Our study substantiates the utility of the Complement Multiplex assay as a tool for measuring complement activation and deficiencies. Quantifying complement cleavage fragments in patients exhibiting classical or alternative pathway activation allowed evaluating the activation state of the whole cascade.

Background: Neurofibromatosis type 1 (NF1) is characterized by the highly variable and unpredictable development of benign peripheral nerve sheath tumors named cutaneous (cNFs), subcutaneous (scNFs), and plexiform (pNFs) neurofibromas.Objectives: A significant genetic component in the variability of neurofibroma incidence was evidenced, but without the influence of the causative NF1 gene pathogenic variant. To identify neurofibroma modifier genes, a NF1 patient database was developed.Methods: All patients were phenotypically evaluated by a medical practitioner using a standardized questionnaire and the causal NF1 variant identified. We enrolled 1,333 NF1 patients who were genotyped for more than 7 million common variants.Results: Genome-wide association case-only study identified a significant association in 9q21.33 for the pNFs phenotype in the discovery cohort. Twelve, three, and four regions suggestive of association at the 10-6 threshold were identified for pNFs, cNFs, and scNFs, respectively. Evidence of replication was observed for four, two, and six loci, including 168 candidate modifier protein-coding genes. Among the candidate modifier genes, some were implicated in the RAS-MAPK pathway, cell cycle control, and myelination. Using an original CRISPR/Cas9-based functional assay, we confirmed GAS1 and SPRED2 as pNFs and scNFs candidate modifiers, as their inactivation specifically affected NF1-mutant Schwann cells growth.Conclusion: Our study may shed new light on the pathogenesis of NF1-associated neurofibromas and will hopefully contribute to the development of personalized care for this deleterious and life-threatening condition.

BACKGROUND:During a pandemic like COVID-19, hospital resources are constrained and accurate severity triage of the patients is required. OBJECTIVE:The objective of this study is to estimate the predictive performances of candidate biomarkers for short-term worsening (STW) of COVID-19. DESIGN:Prospective, multicenter (20 hospitals in Paris) cohort study of consecutive COVID-19 patients with systematic biobanking at admission, during the first waves of COVID-19 in France in 2020 (COVIDeF cohort). SETTING AND PARTICIPANTS:Consecutive COVID-19 patients were screened for inclusion. They were excluded in presence of severity criteria defined by either an ICU admission, mechanical ventilation (including noninvasive ventilation), acute respiratory distress, or in-hospital death before sampling. Routine blood tests measured during usual care and centralized systematic measurement of creatine kinase, C-reactive protein (CRP), procalcitonin, soluble urokinase plasminogen activator receptor (suPAR), high-sensitive troponin T (TnT-hs), N terminal pro-B natriuretic peptide (NT-proBNP), calprotectin, platelet factor 4, mid-regional pro-adrenomedullin (MR-proADM), and proendothelin were performed. OUTCOME MEASURES AND ANALYSES:The primary outcome was STW, defined by a severity criteria within 7 days. A backward stepwise logistic regression model and a 'best subset' approach were used to identify independent association, and the area under the receiving operator characteristics (AUROC) was computed. RESULTS:Five hundred and eleven patients were analyzed, of whom 60 (11.7%) experienced STW. Median time to occurrence of a severity criteria was 3 days. At admission, lower values of eosinophils, lymphocytes, platelets, alanine aminotransferase, and higher values of neutrophils, creatinine, urea, CRP, TnT-hs, suPAR, NT-proBNP, calprotectin, procalcitonin, MR-proADM, and proendothelin were predictive of worsening. Stepwise logistic regression identified three biomarkers significantly associated with worsening: CRP [adjusted odds ratio (aOR): 1.10, 95% confidence interval (95% CI): 1.06-1.15 for a 10-unit increase, AUROC: 0.73 (0.66-0.79)], procalcitonin [aOR: 0.42, 95% CI: 0.22-0.81, AUROC: 0.69 (0.64-0.88)], and MR-proADM [aOR: 2.85, 95% CI: 1.74-4.69, AUROC: 0.75 (0.69-0.81)]. These biomarkers outperformed clinical variables except diabetes and cancer comorbidities. CONCLUSION:In this multicenter prospective study that assessed a large panel of biomarkers for COVID-19 patients, CRP, procalcitonin, and MR-proADM were independently associated with the risk of STW. TRIAL REGISTRATION:ClinicalTrials.gov NCT04352348.

Neurofibromatosis type-1 is a genetic disorder caused by loss-of-function variants in the tumor-suppressor NF1. Approximately 4% to 11% of neurofibromatosis type-1 patients have a NF1 locus complete deletion resulting from nonallelic homologous recombination between low copy repeats. Codeleted genes probably account for the more severe phenotype observed in NF1-deleted patients. This genotype-phenotype correlation highlights the need for a detailed molecular description. A droplet digital PCR (ddPCR) set along the NF1 locus was designed to delimitate the three recurrent NF1 deletion breakpoints. The ddPCR was tested in 121 samples from nonrelated NF1-deleted patients. Classification based on ddPCR versus multiplex ligation-dependent probe amplification (MLPA) was compared. In addition, microsatellites were analyzed to identify parental origin of deletions. ddPCR identified 77 type-1 (64%), 20 type-2 (16%), 7 type-3 (6%), and 17 atypical deletions (14%). The results were comparable with MLPA, except for three atypical deletions misclassified as type-2 using MLPA, for which the SUZ12 gene was not deleted. A significant maternal bias (25 of 30) in the origin of deletions was identified. This study proposes a fast and efficient ddPCR quantification to allow fine NF1 deletion classification. It indicates that ddPCR can be implemented easily into routine diagnosis to complement the techniques dedicated to NF1 point variant identification. This new tool may help unravel the genetic basis conditioning phenotypic variability in NF1-deleted patients and offer tailored genetic counseling.